Identification of an agonist binding site motif that regulates biased signaling of GPCRs through altered conformations of intracellular loop 2

We recently identified a signaling‐biased agonist of the D2 dopamine receptor (D2R), MLS1547, that stimulates G protein‐mediated signaling, but is ineffective in recruiting β‐arrestin. MLS1547 was found to uniquely interact with a hydrophobic binding pocket formed by residues I184, F189, and V190 at the interface between the fifth transmembrane segment (TM5) and the second extracellular loop (EL2) of the D2R. Detailed investigations on the role of these specific residues lead to the identification of F189 (residue 5.38 using Ballesteros‐Weinstein numbering) as a micro‐switch for regulating D2R interactions with β‐arrestin. As position 5.38 is relatively conserved (frequently Phe or Tyr) in class A GPCRs, we constructed alanine mutations (Phe/Tyr to Ala) at this position within all D 2 ‐like receptors (D2R, D3R, and D4R), the β 2 ‐adrenergic receptor (β2R) and the V 2 vasopressin receptor (V2R). Strikingly, we found that the alanine 5.38 mutations negated the receptors' ability to recruit β‐arrestin in response to agonists, while G protein‐signaling efficacy was maintained. These data suggest that the presence of a Phe or Tyr residue at position 5.38 in these GPCRs is critical for stabilizing an activate state for recruiting β‐arrestin. To investigate how alterations at this position produce conformational rearrangements resulting in signaling bias, we used the β2R, for which active state crystal structures are available, to build both β2R‐WT and β2R‐Y199A models in complex with the full agonist BI‐167107, and performed extensive molecular dynamics simulations. Using this approach, we identified residues that differentially interact with BI‐167107 in the β2R‐WT vs. β2R‐Y199A leading to conformational rearrangements that propagate through the TM3‐TM4‐TM5 interface to the intracellular side of the receptor. These coordinated changes result in a different tilt of TM4, face shift of TM4 and TM5 on their extracellular sides, and an altered orientation of IL2 in the β2R‐Y199A compared to the β2R‐WT. Strikingly, such coordinated changes and altered IL2 conformations are reminiscent of the differences between the recently solved cryo‐EM structure of the rhodopsin‐Gi complex and the crystal structure of the rhodopsin‐β‐arrestin complex. These results describe a structural basis for how ligand binding site alterations can modulate GPCR coupling to different transducers resulting in biased signaling. Support or Funding Information NINDS Intramural Research Program This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .